Relative Quantitation of Glycoisoforms of Intact Apolipoprotein C3 in Human Plasma by Liquid Chromatography–High-Resolution Mass Spectrometry

Abstract

Glycosylation is one of the most important post-translational modifications to mammalian proteins. Distribution of different glycoisoforms of certain proteins may reflect disease conditions and, therefore, can potentially be utilized as biomarkers. Apolipoprotein C3 (ApoC3) is one of the many plasma glycoproteins extensively studied for association with disease states. ApoC3 exists in three main glycoisoforms, including ApoC3-1 and ApoC3-2, which contain an O-linked carbohydrate moiety consisting of three and four monosaccharide residues, respectively, and ApoC3-0 that lacks the entire glycosylation chain. Changes in the ratio of different glycoisoforms of ApoC3 have been observed in pathological conditions such as kidney disease, liver disease, and diabetes. They may provide important information for diagnosis, prognosis, and evaluation of therapeutic response for metabolic conditions. In this current work, a liquid chromatography (LC)-high-resolution (HR) time-of-flight (TOF) mass spectrometry (MS) method was developed for relative quantitation of different glycoisoforms of intact ApoC3 in human plasma. The samples were processed using a solid-phase extraction (SPE) method and then subjected to LC-full scan HRMS analysis. Isotope peaks for each targeted glycoisoform at two charge states were extracted using a window of 50 mDa and integrated into a chromatographic peak. The peak area ratios of ApoC3-1/ApoC3-0 and ApoC3-2/ApoC3-0 were calculated and evaluated for assay performance. The results indicated that the ratio can be determined with excellent reproducibility in multiple subjects. It has also been observed that the ratios remained constant in plasma exposed to room temperature, freeze-thaw cycles, and long-term frozen storage. The method was applied in preliminary biomarker research of diabetes by analyzing plasma samples collected from normal, prediabetic, and diabetic subjects. Significant differences were revealed in the ApoC3-1/ApoC3-0 ratio and in the ApoC3-2/ApoC3-0 ratio among the three groups. The workflow of intact protein analysis using full scan HRMS established in this current work can be potentially extended to relative quantitation of other glycosylated proteins. To our best knowledge, this is the first time that a systematic approach of relative quantitation of targeted intact protein glycoisoforms using LC-MS has been established and utilized in biomarker research.

Structure of HDL: Particle Subclasses and Molecular Components

“…This is then linked to reduced inhibition of lipoprotein lipase and improved triglyceride clearance. In line, the production rate of apoC-III 1 and -III 2 is more strongly correlated with plasma triglyceride levels than apoC-III 0 (Mauger et al 2006), increased apoC-III 1 / apoC-III 0 ratio has been found in diabetic subjects (Jian et al 2013), and HDL3 from subjects with low HDL-C is characterised by higher levels of monosialylated apoC-III than subjects with high HDL-C (Mazur et al 2010). The evaluation of apoC-III isoforms is complicated by the fact that apoC-III 0 can be separated into a non-glycosylated form and glycosylated but non-sialylated forms (Bruneel et al 2008;Holleboom et al 2011;Nicolardi et al 2013a).…”

Internal Standards for Absolute Quantification of Large Molecules (Proteins) from Biological Matrices by LC-MS/MS

“…Solid phase extraction (SPE) is another purification technique that is employed solely or along with other purification techniques for sample clean up wherein the analyte is a smaller protein or peptide [12,[14][15][16][17]. Several mixed mode SPE cartridges, combining reversed phase stationary phase along with strong cation exchange or weak anion exchange, are commercially available for peptide analysis.…”

“…Columns with larger pore stationary phases (300 Å) allow improved penetration of larger molecules as well as the use of higher flow rates with reduced band broadening, and therefore provide greater selectivity for some peptides and proteins. Two-dimensional chromatographic separations, such as ion-exchange chromatography (IEC)-RPLC or RPLC-HILIC, have been utilized to fractionate and clean up samples, thus improving sensitivity of detection [16,24,46,52,53]. Additionally, many reported methods have used column trapping prior to analytical separation.…”

“…The complex structure consisting of specific intramolecular folding of amino acid chains as well as intramolecular di-sulfide linkages and presence of post translational modifications makes it difficult to synthesize these proteins in a reproducible manner. If the analyte protein is small, a SIL form of the protein can be chemically synthesized using solid-phase synthesis [16,56]. However, for most protein analytes, the production of its isotopic labeled form requires a cellular environment.…”

Development of multiplex mass spectrometric immunoassay for detection and quantification of apolipoproteins C-I, C-II, C-III and their proteoforms

“…Importantly, several proteoforms of apoCs exist [32] which reflect their function in vivo . MS-based methods provide a unique opportunity to study apoCs proteoforms [33–37], and correlate their structural modifications with function in physiological and pathological states.…”